Elevated precursor 16S rRNA levels suggest the presence of growth inhibitors in wastewater

Conventional activated sludge systems require bacteria to grow to avoid washout through decay and routine solids wasting. Recently we developed a procedure targeting precursor 16S ribosomal RNA to measure the in situ growth activity of phylogenetically defined microbial populations, and this procedure was used to study the growth of bacteria in activated sludge systems. The current study significantly expands this previous work by quantifying levels of precursor 16S ribosomal RNA within individual cells of pure cultures of bacteria exposed to various culture conditions. Initially, three ranges (Type I, Type II, and Type III) of precursor 16S ribosomal RNA levels were defined by whole cell fluorescence in situ hybridization of a pure culture of Acinetobacter calcoaceticusT prepared in three culture conditions. Low levels of precursor 16S ribosomal RNA (Type I) corresponded to a stationary phase culture prepared overnight in Luria-Bertani medium. Intermediate levels of precursor 16S ribosomal RNA (Type II) corresponded to a culture transferred into fresh Luria-Bertani medium, and high levels of precursor 16S ribosomal RNA (Type III) corresponded to a culture treated with the growth inhibiting antibiotic chloramphenicol. Subsequently, the abundance of individual cells of each Type were measured in four different pure cultures after exposure to 0.45-microm filtered primary effluent collected from four different conventional activated sludge treatment plants in Cincinnati, OH, USA. Individual cells of each Type were observed in the culture of A. calcoaceticusT exposed to each of the four primary effluents. Only Type I cells were observed in cultures of A. johnsoniiT, A. johnsonii strain 210a, and Escherichia coliT exposed to each of the four primary effluents. These results suggest that the growth of A. calcoaceticusT was inhibited by an unidentified component of filtered primary effluent present in each of the four wastewaters; whereas the growth of A. johnsoniiT, A. johnsonii strain 210a, and E. coliT were not inhibited. These results have significance for understanding the growth of phylogenetically defined microbial populations within activated sludge treatment systems. If the pattern of elevated p16S rRNA levels observed in A. calcoaceticusT is prevalent in many microbial populations in activated sludge systems, this may have implications for preventing washout of critical microbial populations that may be experiencing growth inhibition.